Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing alpha or beta amino acid or substituted amino acid...
Reexamination Certificate
2001-10-11
2003-12-02
Saucier, Sandra E. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing alpha or beta amino acid or substituted amino acid...
C435S107000, C435S108000, C435S109000, C435S110000, C435S113000, C435S114000, C435S116000
Reexamination Certificate
active
06656710
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims priority to German Application DE 10050123.0, filed Oct. 11, 2000. The entire contents of the application is incorporated herein by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the processes of racemization and deprotection of special N-protected amino acids in the acylase/racemase system for the total conversion of special N-protected racemic amino acids into optically pure amino acids.
2. Discussion of Background
Optically pure enantiomers of both L- and D-amino acids are important starting compounds in chemical syntheses, as well as for parenteral nutrition. Many methods of producing oprtically pure amino acids are possible and known to a person skilled in the art. Suitable processes include relevant enzymatic processes since they take place catalytically and produce with very high enantiomer concentrations of amino acids.
A racemic mixture of amino acids is not optically pure and contains both L-amino acid and D-amino acid enantiomers. Both L-amino acid and D-amino acid enantiomers can be utilized through enzymatic catalysis of a racemic mixture of N-protected amino acids. For example, it is known that L-amino acids are prepared from a racemic mixture of N-acetylated amino acids by using amino acid acylases. However, it is thought that these acylases are specific only for the cleavage of N-acetyl-protected amino acids and amines/alcohols (EP99118844.2; A. S. Bommarius et al., Tetrahedron; Asymmetry, 1997, Vol. 8, 3197-3200). Further, various racemization processes have been developed to prepare L-amino acids from the remaining D-acetyl amino acid fraction of the racemic mixture. DE 19935268.2 discloses an acetylamino acid racemase in the acylase/acetylamino acid racemase system that can prepare optically pure L-methionine from a racemic mixture of N-acetylmethionine.
Less specific N-acetylamino acid racemases (AAR) have been described previously. These racemases can be found in the microorganisms
Streptomyces atratus
Y-53 (Tokuyama et al., Appl. Microbiol. Biotechnol. 1994, 40, 835-840) and
Amycolatopis
sp. TS-1-60 (Tokuyama et al., Appl. Microbiol. Biotechnol. 1995a, 42, 853-859). For example, the racemase of Amycolatopis sp. TS-1-60 can catalyze the racemization of N-carbamoylamino acids to L-amino acids, although with less than optimal activity.
Processes for complete conversion of amino acids other than acetyl-protected or carbamoyl-protected amino acids to optically enriched amino acids are not known. Further, racemases with the ability to convert amino acids other than acetyl-protected or carbamoyl-protected amino acids are not known. Therefore, there is a need for enzymatic processes that racemize N-protected amino acids in general, as well as those N-protected amino acids other than acetyl-protected or carbamoyl-protected amino acids. The N-protected amino acid products of such racemic converstions may then be converted into the optically enriched amino acid by a subsequent enzymatic cleavage of the protecting group(s).
SUMMARY OF THE INVENTION
One object of the present invention is a process for the racemization of N-protected amino acids, comprising contacting a compound of the general formula (I):
wherein
X=O, NH,
R
1
=CH
3
, CH
3
CH
2
, tert-butyl, benzyl and R
2
denotes the &agr;-radical of a natural or synthetic amino acid, with an N-acetylamino acid racemase.
Another object of the present invention is a process for the cleavage of the protective group from N-protected amino acids, comprising contacting a compound of the general formula (I):
wherein
X=O, NH,
R
1
=CH
3
, CH
3
CH
2
, tert.-butyl, benzyl, wherein if X is NH, then R
1
may be H, and R
2
denotes the &agr;-radical of a natural or synthetic amino acid, with an amino acid acylase.
Another object of the present invention is a process, comprising contacting a compound of the general formula (I):
wherein
X=O, NH,
R
1
=CH
3
, CH
3
CH
2
, tert-butyl, benzyl, wherein if X is NH, then R
1
may be H, and
R
2
denotes the &agr;-radical of a natural or synthetic amino acid,
with an N-acetylamino acid racemase (AAR) in the presence of an amino acid acylase. In one embodiment, the racemase contacts the compound first followed by the acylase. Either or both the racemase and the acylase may be in a homogeneous free form, a recombinant free form, a part of a host organism, a portion of a digested cell mass, an immobilised form.
Another object of the present invention is to provide a process for the production of optically enriched amino acids from a racemic mixture of amino acids that are N-protected.
Another object of the present invention is to provide a process for the production of optically enriched amino acids from a racemic mixture of amino acids that are N-protected by means of a urethane-protected or carbamoyl-protected amino acid.
DETAILED DESCRIPTION OF THE INVENTION
Unless specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled artisan of molecular biology and biochemistry.
Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting.
Reference is made to standard textbooks of molecular biology that contain definitions and methods and means for carrying out basic scientific techniques, encompassed by the present invention. See, for example, Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1982) and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989) and various references cited therein.
The term “&agr;-radical of an amino acid” is understood to denote the radical located on the &agr;-C atom of an &agr;-amino acid. This radical may be derived from a natural amino acid, as described in Beyer-Walter, Lehrbuch der organischen Chemie, S. Hirzel Verlag Stuttgart, 22
nd
Edition, 1991, p. 822f. Furthermore, corresponding &agr;-radicals of synthetic &agr;-amino acids are also covered, as listed for example in DE19903268.8.
“Optically enriched”, or “enantiomer-enriched”, compounds within the scope of the present invention is understood to mean the presence of an optical antipode mixed with the other antipodes in a concentration of >50 mole %.
“Polynucleotide” in general relates to polyribonucleotides and polydeoxyribonucleotides, it being possible for these to be non-modified RNA or DNA or modified RNA or DNA.
“Polypeptides” are understood as meaning peptides or proteins, which comprise two or more amino acids, bonded via peptide bonds.
“Free form” of a polynucleotide or polypeptide refers to a polynucleotide or polypeptide separated out of its natural environment and into an aqueous solution.
“Digested cell mass” is any solution of cellular components produced as a direct result of disrupting the integrity of a cell wall and/or cell membrane. The cell may be a unicellular organism and/or may be a portion of a multi-cellular organism.
“N-acetylamino acid racemase” denotes a class of enzymes that can racemise optically enriched N-acetylamino acids. On account of the great similarity to one another, all N-acetylamino acid racemases known to the person skilled in the art can be used for the present conversions. Preferably, the racemases to be used are those from
Streptomyces atratus
Y-53 as well as Amycolatopis sp. TS-1-60. A process that is particularly preferred is one comprising the N-acetylamino acid racemase from
Amycolatopsis orientalis
subspecies
lurida
(SEQ ID NO. 2), since this particular N-acetylamino ac
Bommarius Andreas
Drauz Karlheinz
Verseck Stefan
Degussa - AG
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Saucier Sandra E.
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